Identification of novel glycosyltransferases required for assembly of the Pasteurella multocida A:1 lipopolysaccharide and their involvement in virulence

Abstract

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel alpha-1,6 glucosyltransferase, a beta-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive.

FIG. 1.

Structures of the LPS molecules expressed by P. multocida strains Pm70 (A) and VP161 (B) and the glycoform C observed only in the VP161 hptE mutant (C). Two inner-core LPS glycoforms were observed in Pm70 and VP161 wild-type strains, and the variable sections are boxed in panel B and designated glycoform A and B. The gene known (B) or predicted (A and C) to be required for each biosynthetic step in the synthesis of each molecule is shown below the linkages. The residues are Glc, glucose; Hep, heptose; Gal, galactose; GlcNAc, N-acetylglucosamine; and PEtn, phosphoethanolamine.

FIG. 2.

Anomeric regions of the ¹H-NMR spectra of core oligosaccharide from the VP161 wild-type parent strain (a) and the gctA mutant AL574 (b). The anomeric resonances are as indicated. The asterisks refer to heterogeneity in the parent strain caused by the variable presence of the α-glucose residue, which affects chemical shifts of the marked anomeric resonances and which is absent in the mutant strain, where the α-glucose residue is absent. The insets show the structures for the wild-type and mutant strains. The spectra were recorded in D₂O at 25°C and referenced to the deuterated water signal at 4.78 ppm.

FIG. 3.

Sensitivities of P. multocida strains to killing by the action of fowlicidin 1. The MIC of fowlicidin 1 was determined by growth of each strain in 50% BHI containing increasing concentrations of fowlicidin 1. The strains tested were the parent (AL435), PM0442 mutant (strain AL669 with an insertion in a gene unrelated to LPS biosynthesis), pcgC mutant (PCho⁻ strain [14]), gatA mutant (AL725), hptE mutant (AL523), gctB mutant (AL554), gctA mutant (AL574), and hptC mutant (AL696). The MIC was determined by averaging the concentration of fowlicidin 1 that completely inhibited bacterial growth and the next-lowest concentration that allowed some growth. Standard deviations were calculated assuming that the MIC for each assay was equally likely to lie at any value within this interval. The reported values are the MICs for three replicates, and the error bars show ±1 standard deviation.